JP6537076B2 - Secretion signal peptide and protein secretion and cell surface display using the same - Google Patents

Description

  The present invention relates to a secretion signal peptide and the secretion and cell surface display of a protein utilizing the same.

  In recent years, from the viewpoint of realizing a low carbon recycling society, it is desired to introduce a technology for producing ethanol from lignocellulosic biomass which is a renewable resource. In order to disseminate it, it is urgently needed to construct an efficient, low-cost simultaneous saccharification and fermentation process by integrating multi-step processes for converting plant raw materials into ethanol.

  In this construction, for example, yeast having a saccharification ability is produced by presenting an enzyme such as cellulase or hemicellulase on the cell surface. Cell surface display techniques include methods using GPI anchor proteins of cell surface localized proteins. Such cell surface display technology uses, for example, an expression cassette containing a promoter, a secretion signal peptide, a gene for surface display, and a gene of GPI anchor protein.

  There are various reports on GPI anchor proteins and promoters (patent documents 1 to 4 and non-patent documents 1 to 5).

  A secretory signal peptide is a peptide that is localized at the cell membrane, cell wall, or at the N-terminus of a protein secreted extracellularly. In the secretory expression system of protein, it has been reported that the expression efficiency and the expression success rate can be raised by replacing the secretory signal peptide of the target protein with a highly efficient secretory signal peptide. Therefore, high efficiency of the secretion signal peptide is an important factor in enhancing the cell surface display system of proteins and the secretory production system.

  As a typical secretory signal peptide derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) used in a highly efficient secretory protein expression system in existing yeast, for example, a secretory signal peptide derived from α-factor (non-patent document 6) (MFα prepro). In addition, a secretion signal peptide derived from Saccharomyces cerevisiae, which is considered to have a secretory ability exceeding that of this α-factor-derived secretion signal peptide, has also been reported (Patent Document 5).

  On the other hand, in general, it is known that the secretory ability of a secretory signal peptide largely fluctuates depending on the combination with a promoter and a protein connected thereto. Moreover, it is difficult at present to predict the stability of secretory capacity from secretory signal peptide sequences. Therefore, even in the case of a secretory signal sequence capable of exerting high secretory ability in combination with a specific promoter and protein, it may frequently occur that the secretory ability is greatly reduced in other combinations.

  For example, the ability to secrete a secretory signal peptide, which is considered to be highly efficient in Patent Document 5, is evaluated using only the activity when combined with a single promoter (HSP1 promoter) and a single protein (luciferase) as an index. It is.

  Furthermore, the ability to secrete a secretory signal peptide, which is considered to be highly efficient in Patent Document 5, is evaluated using only the activity when the target protein is secreted extracellularly (culture supernatant) as an index.

JP, 2011-160727, A JP 2008-86310 A JP 2007-189909 A JP 2005-245335 A JP, 2007-167062, A JP, 2011-30563, A

Biotechnol. Lett., 2010, 32, 1131-1136. Mol. Microbiol., 2004, 52, 1413-1425. Appl. Environmen. Microbiol., 1997, 63, 615-620. Biotechnol. Lett., 2010, 32, pp. 255-260. J. Bacteriol., 1997, 179, 1513-1520. Protein Eng., 1996, Vol. 9, pages 1055-1065. Appl. Microbiol. Biotechnol., 2006, 72, 1136-1143. Nature Methods, 2009, 6, 343-345. FEMS Yeast Res., 2014, 14: 399-411 Biotechnol. Biofuels, 2014, Volume 7, Page 8 Enzyme Microb. Technol., 2012, 50, 343-347 J Biochem., 2012, 145, 701-708.

  An object of the present invention is to provide a secretory signal peptide having a secretory ability exceeding that of a conventionally used secretory signal peptide in secretory production of proteins and cell surface display.

  Another object of the present invention is to provide a secretory signal peptide which stably has a secretory ability higher than that of conventionally used secretory signal peptides even in combination with a plurality of different promoters and proteins.

The present invention provides an expression vector comprising the following (i), (ii) and (iii) (this expression vector is also referred to as a secretory expression vector):
(I) promoter DNA,
(Ii) the following:
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2,
(B) a peptide having an amino acid sequence having at least 70% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having a secretory signal activity,
(C) A peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2 and having a secretory signal activity,
(D) A peptide encoded by a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence represented by SEQ ID NO: 1 and comprising a peptide having secretory signal activity, and (e) a nucleotide sequence represented by SEQ ID NO: 1 A peptide encoded by a nucleotide sequence that hybridizes to a complementary strand of DNA and having secretory signal activity,
DNA encoding any peptide selected from the group consisting of: (iii) DNA encoding the target protein, or a cloning site for inserting DNA encoding the target protein.

  In one embodiment, in the secretory expression vector, the promoter is a SED1 promoter.

  In one embodiment, in the secretory expression vector, the (iii) is a DNA encoding a target protein.

  The present invention provides a transformed yeast into which the above secretion type expression vector has been introduced.

The present invention provides a method for producing a yeast that secretes and produces a protein, said method comprising
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, (b) an amino acid sequence having at least 70% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, secretion signal activity (C) a peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2 and having secretory signal activity (D) a peptide having a secretory signal activity which is encoded by a base sequence having at least 70% sequence identity to the base sequence represented by SEQ ID NO: 1, and (e) from the base sequence represented by SEQ ID NO: 1 Selected from the group consisting of peptides having a secretory signal activity, encoded by a base sequence that hybridizes to the complementary strand of the DNA DNA encoding the peptide of Zureka; and an expression cassette comprising a DNA encoding a desired protein is introduced into yeast, including the step of obtaining the transformed yeast.

  The present invention provides a method for secretory production of a protein in yeast, comprising the step of culturing the above-mentioned transformed yeast or the yeast produced by the above-mentioned method.

The present invention provides an expression vector comprising the following (i), (ii), (iii) and (iv) (this expression vector is also referred to as a surface display type expression vector):
(I) promoter DNA,
(Ii) the following:
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide having an amino acid sequence having at least 70% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having a secretory signal activity,
(C) A peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2 and having a secretory signal activity,
(D) A peptide encoded by a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence represented by SEQ ID NO: 1 and comprising a peptide having secretory signal activity, and (e) a nucleotide sequence represented by SEQ ID NO: 1 A peptide encoded by a nucleotide sequence that hybridizes to a complementary strand of DNA and having secretory signal activity,
DNA encoding any peptide selected from the group consisting of
(Iii) a DNA encoding a target protein, or a cloning site for inserting a DNA encoding the target protein;
(Iv) DNA encoding an anchor domain.

  In one embodiment, in the surface layer display type expression vector, the promoter is a SED1 promoter.

  In one embodiment, in the surface display type expression vector, the anchor domain is a SED1 anchor domain.

  In one embodiment, in the surface layer display type expression vector, the above (iii) is a DNA encoding a target protein.

  The present invention provides a transformed yeast into which the surface display type expression vector has been introduced.

The present invention provides a method for producing a yeast for surface display of a protein, said method comprising
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, (b) an amino acid sequence having at least 70% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, secretion signal activity (C) a peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2 and having secretory signal activity (D) a peptide having a secretory signal activity which is encoded by a base sequence having at least 70% sequence identity to the base sequence represented by SEQ ID NO: 1, and (e) from the base sequence represented by SEQ ID NO: 1 Selected from the group consisting of peptides having a secretory signal activity, encoded by a base sequence that hybridizes to the complementary strand of the DNA Step and an expression cassette comprising a DNA encoding an anchor domain was introduced into yeast, obtaining a transformed yeast,; DNA encoding a protein of interest; DNA encoding a peptide of Zureka
including.

  According to the present invention, proteins can be secreted with high activity or presented on the cell surface. The secretion signal peptide of the present invention can be suitably used for both protein secretion and cell surface display. Furthermore, the secretion signal peptide of the present invention can stably provide high secretion ability in combination with various promoters and protein genes.

It is a graph which shows a time-dependent change of (beta) -glucosidase activity of the microbial cell at the time of culture | cultivation about the various surface layer display transformation yeast obtained using expression cassette X1-X4. It is a graph which shows a time-dependent change of (beta) -glucosidase activity in the culture medium at the time of culture | cultivation about the various secretory transformed yeasts obtained using expression cassette X5-X8. It is a graph which shows the endoglucanase activity of the microbial cell after culture for 48 hours about the various surface layer display transformation yeast obtained using expression cassette X9-X11. It is a graph which shows a time-dependent change of (beta) -glucosidase activity of the microbial cell at the time of culture | cultivation about the various surface layer display transformation yeast obtained using expression cassette X12-X17. It is a graph which shows the time-dependent change of (beta) -glucosidase activity in the culture medium at the time of culture | cultivation about the various secretory transformed yeasts obtained using expression cassette X18 and X19. It is a graph which shows the relative value of the GFP fluorescence intensity in the culture medium after culture for 24 hours about various secretory transformation yeasts obtained using expression cassette X20-X22.

  The present invention will be described in detail below.

(Secretory signal peptide)
Secretory signal peptides are peptides that are usually bound to the cell membrane, cell wall, or are usually bound to the N-terminus of proteins secreted out of the cell membrane. Secretory signal peptides are usually removed by being degraded by signal peptidases in the process of secretion proteins from inside the cell across the cell membrane and being secreted out of the cell. For example, a secretory signal peptide is linked to the protein having secretory signal peptide (secretory protein) or the N-terminus of a heterologous protein in the cell in which these proteins are to be expressed, or the secretory protein or It functions to secrete heterologous proteins extracellularly.

  In the present specification, in yeast, functioning as a secretory signal peptide upon expression of a target protein is also referred to as “secretory signal activity”.

According to the present invention, any peptide selected from the group consisting of the following (a) to (e) is provided as a secretion signal peptide:
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide having an amino acid sequence having at least 70% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and having secretory signal activity;
(C) a peptide having an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2 and having secretory signal activity;
(D) a peptide encoded by a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence represented by SEQ ID NO: 1 and comprising a peptide having a secretory signal activity; and (e) a nucleotide sequence represented by SEQ ID NO: 1 A peptide encoded by a nucleotide sequence that hybridizes to a complementary strand of DNA and having secretory signal activity.

  Furthermore, according to the present invention, a DNA encoding any peptide selected from the group consisting of the above (a) to (e) (herein also simply referred to as "the secretion signal peptide of the present invention"). Also provided is a polynucleotide comprising: One example of a DNA encoding the secretion signal peptide of the present invention is the base sequence represented by SEQ ID NO: 1, which encodes the amino acid sequence represented by SEQ ID NO: 2.

  The amino acid sequence represented by SEQ ID NO: 2 and the base sequence encoding it (SEQ ID NO: 1) are secretory signal peptides of SED1, which is a major cell surface localized protein in stationary phase of the yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae). Although it originates, an origin is not limited to this. SED1 is induced by stress and is thought to contribute to the maintenance of cell wall integrity. The gene (Sed1) of SED1 can be obtained, for example, based on the sequence information registered in GenBank, using methods commonly used by those skilled in the art (GenBank accession number NM_001180385; NCBI Gene ID: 851649).

  Here, the protein or peptide described herein consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the disclosed amino acid sequence, and a desired function or effect in the present invention It may be a substantially protein or peptide, and a polynucleotide may include one encoding such a protein or peptide. Mutation (for example, deletion, substitution or addition) of an amino acid to the disclosed amino acid sequence may be any one type, or two or more types may be combined. Also, the total number of these mutations is one or several, but is not particularly limited as long as it substantially has its desired function or effect, and may also depend on the size of the protein or peptide. The total number of mutations is, for example, 1 or more and 10 or less, 1 or more and 5 or less, 1 or more and 4 or less, 1 or more and 3 or less, or 1 or more and 2 or less, but is not limited thereto . Also, the total number of mutations may, for example, be in the range satisfying the sequence identity described below. Examples of amino acid substitution may be any substitution as long as each function or effect is substantially retained. For example, conservative substitutions can be mentioned. Conservative substitutions specifically include substitutions within the following groups (ie, between the amino acids shown in parentheses): (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid) ), (Asparagine, glutamine), (serine, threonine), (lysine, arginine), (phenylalanine, tyrosine).

  In other embodiments, the proteins or peptides described herein have an amino acid sequence having, for example, 70% or more sequence identity to the disclosed amino acid sequence, and desired in the present invention The protein or peptide may have substantially the function or effect of and the polynucleotide may encode such a protein or peptide. Sequence identity in the amino acid sequence may also be 74% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more.

  As used herein, sequence identity or similarity is the relationship between two or more proteins or two or more polynucleotides as determined by comparing the sequences, as known in the art. . Sequence "identity" is determined between the protein or polynucleotide sequences, as determined by the alignment between the protein or polynucleotide sequences, or, in some cases, by the alignment between partial sequences of stretches. It means the degree of sequence invariance. Also, "similarity" is between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases, by alignment between partial sequences of stretches. It means the degree of correlation. More specifically, it is determined by sequence identity and conservation (substitutions that maintain physiochemical properties at particular amino acids or sequences in the sequence). The similarity is referred to as "Similarity" in the sequence homology search results of BLAST described later. Preferably, the method of determining identity and similarity is a method designed to provide the longest alignment between the sequences being compared. Methods to determine identity and similarity are provided as programs available to the public. For example, the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; Altschyl et al., Nucleic Acids Res., 1997, 25: 3389-3402. ) Can be used. Conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

  In yet another embodiment, the protein or peptide described herein may be encoded by DNA hybridized with DNA consisting of a nucleotide sequence complementary to DNA consisting of the disclosed nucleotide sequence Well, and polynucleotides may include such hybridized DNA. The hybridization between the DNA consisting of the disclosed base sequence and the DNA consisting of the complementary base sequence is preferably carried out under stringent conditions.

  Stringent conditions mean, for example, conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a nucleic acid having high identity of the base sequence, ie, 70% or more, 75% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 92% or more, 95% or more Conditions under which the complementary strand of DNA consisting of a nucleotide sequence having 98% or more or 99% or more identity hybridizes and the complementary strand of nucleic acid having a lower homology than that does not hybridize can be mentioned. More specifically, the sodium salt concentration is, for example, 15 mM to 750 mM, 50 mM to 750 mM, or 300 mM to 750 mM, the temperature is, for example, 25 ° C. to 70 ° C., 50 ° C. to 70 ° C., or 55 ° C. to 65 ° C., and formamide The condition is, for example, 0% to 50%, 20% to 50%, or 35% to 45%. Furthermore, under stringent conditions, washing conditions for the post-hybridization filter include sodium salt concentration of, for example, 15 mM to 600 mM, 50 mM to 600 mM, or 300 mM to 600 mM, and temperature of 50 ° C. to 70 ° C., 55 ° C. to 55 ° C. 70 ° C., or 60 ° C. to 65 ° C. Moreover, for example, after hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which the DNA is immobilized, with DNA that hybridizes under stringent conditions. Examples include DNA that can be obtained by washing the filter at 65 ° C. in a 0.1 to 2 × SSC solution (the composition of the 1 × SSC solution is 150 mM NaCl, 15 mM sodium citrate). Hybridization can be performed by a known method such as, for example, the method described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory (2001). The higher the temperature, or the lower the salt concentration, the higher the stringency, and polynucleotides with higher homology (sequence identity) can be isolated.

  In yet another embodiment, the polynucleotide described herein can be combined with the disclosed base sequences with, for example, 70% or more, 75% or more, 78% or more, 80% or more, 85% or more, 90% or more, Examples include polynucleotides having a base sequence having 92% or more, 95% or more, 98% or more, or 99% or more identity, and substantially having the desired function or effect thereof.

  The nucleotide sequence encoding a predetermined amino acid sequence (for example, the amino acid sequence represented by SEQ ID NO: 2) encodes a predetermined amino acid sequence without changing the amino acid sequence of the protein by substitution based on the degeneracy of the genetic code. It is also possible to substitute at least one base of the base sequence with another base. In yet another embodiment, the DNA encoding the secretion signal peptide of the present invention also includes DNA having a nucleotide sequence altered by substitution based on the degeneracy of the genetic code.

  The DNA encoding the secretion signal peptide of the present invention or the polynucleotide containing the DNA is, for example, a predetermined yeast (for example, Saccharomyces cerevisiae) using a primer designed based on the nucleotide sequence shown in SEQ ID NO: 1 The nucleic acid fragment can be obtained as a nucleic acid fragment by performing PCR using, as a template, a DNA extracted therefrom, a nucleic acid derived from various cDNA libraries or a genomic DNA library. A DNA encoding the secretion signal peptide of the present invention or a polynucleotide containing the DNA can be hybridized to a nucleic acid derived from the above library using a probe designed based on the nucleotide sequence shown in SEQ ID NO: 1 By carrying out, it can be obtained as a nucleic acid fragment. The DNA encoding the secretion signal peptide of the present invention or the polynucleotide containing the DNA may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis.

  Furthermore, for the various embodiments described above for the polynucleotide, the DNA encoding the secretion signal peptide of the present invention may be, for example, a DNA consisting of the base sequence represented by SEQ ID NO: 1 according to a conventional mutagenesis method, It can be obtained by modification by a specific mutation method or a molecular evolutionary technique using error prone PCR. Examples of such a method include known methods such as the Kunkel method and the Gapped duplex method, or methods equivalent thereto. For example, a kit for mutagenesis using site-directed mutagenesis (for example, Mutant-K (manufactured by TAKARA), Mutant-G (manufactured by TAKARA), etc.) or the like, or LA PCR in vitro from TAKARA Mutations are introduced using the Mutagenesis series kit.

  As described in detail below, a secretion cassette for secretion production of a target protein and surface layer display of the target protein on the cell surface using a DNA encoding the secretion signal peptide of the present invention or a polynucleotide containing the DNA An expression cassette such as a cassette can be made.

(DNA encoding an anchor domain)
The surface display cassette comprises DNA encoding an anchor domain.

  As the anchor domain, a cell surface localized protein or its cell membrane binding region can be used. "Cell surface localized protein" refers to a protein that is immobilized or attached to or adheres to the cell surface and is localized there. As a cell surface localized protein, a lipid-modified protein is known, and this lipid is immobilized on a cell membrane by covalently binding to a membrane component.

  As a representative example of a cell surface localized protein, GPI (glycosyl phosphoryl inositol: ethanolamine phosphate 6 mannose α-1,2 mannose α-1,6 mannose α-1,4 glucosamine α-1,6 inositol phospholipid) Glycolipid (an anchor protein) used as a basic structure can be mentioned. The GPI anchor protein has GPI which is a glycolipid at its C-terminus, and this GPI is bound to the cell membrane surface by covalently binding to PI (phosphonidyl inositol) in the cell membrane.

  Coupling of GPI to the C-terminus of GPI anchor protein is carried out as follows. The GPI anchor protein is secreted into the endoplasmic reticulum by the action of a secretory signal present at the N-terminal side after transcription and translation. At or near the C-terminus of the GPI anchor protein, there is a region called GPI anchor attachment signal that is recognized when the GPI anchor binds to the GPI anchor protein. In the lumen of the endoplasmic reticulum and the Golgi apparatus, this GPI anchor adhesion signal region is cleaved, and GPI binds to the newly generated C-terminus.

  GPI-bound proteins are transported to the cell membrane by secretory vesicles, and are immobilized on the cell membrane by covalently binding GPI to PI of the cell membrane. Furthermore, the GPI anchor is cleaved by phosphatidylinositol-dependent phospholipase C (PI-PLC), and is presented on the cell surface in a fixed state to the cell wall by being incorporated into the cell wall.

  In the present invention, a polynucleotide encoding the entire cell surface localized protein GPI anchor protein or a region including the cell membrane binding region GPI anchor adhesion signal region can be used. The cell membrane binding region (GPI anchor adhesion signal region) is usually a C-terminal region of cell surface localized protein. The cell membrane binding region may contain a GPI anchor adhesion signal region, and may contain any other part of the GPI anchor protein as long as it does not inhibit the enzyme activity of the fusion protein.

  The GPI anchor protein may be a protein that functions in yeast cells. Such GPI anchor proteins include, for example, α- or a-agglutinin (AGα1, AGA1), TIP1, FLO1, SED1, CWP1 and CWP2. Preferably, SED1 or its cell membrane binding region (GPI anchor adhesion signal region) is used. The base sequence of the anchor protein coding region of Sed1 is shown in SEQ ID NO: 4, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 5 (however, SEQ ID NOs: 4 and 5 represent the initiation codon and the methionine encoded thereby respectively) Not included). The cell membrane binding region (GPI anchor adhesion signal region) of SED1 is, for example, a region including positions 109 to 337 of SEQ ID NO: 5. As the SED1 anchor domain, even if it is the entire length of the amino acid sequence of SEQ ID NO: 5, or a partial sequence thereof (eg, a sequence including the amino acid sequence of positions 109 to 337 of SEQ ID NO: 5) as long as the anchor function is not impaired. ) May be.

  The identity of the sequences described above, hybridization conditions, etc. apply to the anchor domain and its encoding DNA.

  The DNA encoding an anchor domain (eg, cell surface localized protein or its cell membrane binding region) is known as a DNA extracted from a microorganism having these, a nucleic acid derived from various cDNA libraries or genomic DNA libraries as a template, It can be obtained as a nucleic acid fragment by PCR using primers designed based on the sequence information. Such a polynucleotide can be obtained as a nucleic acid fragment by hybridization to a nucleic acid derived from the above library using a probe designed based on known sequence information. It is also possible to cut out and use as a nucleic acid fragment from the existing vector containing the said polynucleotide. The polynucleotides may be synthesized as nucleic acid fragments by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

(Promoter DNA)
The promoter DNA only needs to have promoter activity, and "promoter activity" refers to an activity that causes a transcription factor to bind to the promoter region to initiate transcription. The promoter DNA can be excised from cells, phage or the like carrying a desired promoter region using a restriction enzyme. If necessary, a DNA fragment of the promoter region can be obtained by amplifying a desired promoter region by PCR using primers provided with a restriction enzyme recognition site or an overlapping site with a cloning vector. In addition, desired promoter DNA may be chemically synthesized based on already known nucleotide sequence information of the promoter region.

  The promoter DNA contained in the polynucleotide of the present invention may be any promoter as long as it has promoter activity in yeast. The promoter DNA may be the gene itself for expression or may be derived from another gene. It may also be a promoter of a gene encoding a cell surface localized protein used as an anchor domain. For example, SED1 promoter, TDH3 promoter, PGK1 promoter, CWP2 promoter, and TDH1 promoter can be mentioned. The nucleotide sequences of these promoters are the nucleotide sequences represented by SEQ ID NOs: 3, 12, 13, 18, and 21, respectively. As described above, even if one or two or more (for example, several) nucleotides are mutated, such as deletion, addition or substitution, as described above, as long as the target function is achieved. Good.

(Target protein)
The type of target protein or its source is not particularly limited. Examples of the type of target protein include enzymes, antibodies, ligands, and fluorescent proteins. Examples of the enzyme include cellulolytic enzymes, starch degrading enzymes, glycogen degrading enzymes, xylanolytic enzymes, chitinolytic enzymes, lipolytic enzymes and the like, and more specifically, for example, endoglucanase, cellobiohydrolase, and β-glucosidase, amylase (eg, glucoamylase and α-amylase), lipase and the like can be mentioned.

  The DNA encoding the target protein is preferably a cDNA sequence from which introns have been removed.

  The DNA encoding the target protein may be a sequence encoding the entire length thereof, or may be a sequence encoding a partial region of the target protein as long as it exhibits the activity of the target protein. In addition, as described above, as long as the activity of the target protein is exhibited, a base sequence containing a mutation such as deletion, addition or substitution of one or more (for example, several) nucleotides in the base sequence encoding a naturally occurring protein Or a base sequence encoding a protein consisting of an amino acid sequence containing one or more (for example, several) amino acids including mutations such as deletions, additions or substitutions.

  The DNA (gene) encoding the target protein was designed based on known sequence information, using as a template the DNA extracted from the microorganism having or producing the protein, or various nucleic acid libraries derived from various cDNA libraries or genomic DNA libraries It can be obtained as a nucleic acid fragment by PCR using a primer. Such a polynucleotide can be obtained as a nucleic acid fragment by hybridization to a nucleic acid derived from the above library using a probe designed based on known sequence information. Alternatively, it can be used as a nucleic acid fragment (which may be in the form of an expression cassette) from an existing vector containing a DNA encoding a target protein. Such a gene can also be obtained artificially synthesized based on a sequence optimized in consideration of the host codon frequency as needed.

  Hereinafter, as an example of the target protein, a cellulolytic enzyme will be described as an example.

  Cellulolytic enzymes refer to any enzymes capable of cleaving β1,4-glycosidic bonds. Cellulolytic enzymes may be derived from any cellulose hydrolase-producing bacteria. As the cellulose hydrolase-producing bacteria, typically, Aspergillus (eg, Aspergillus aculeatus, Aspergillus niger, and Aspergillus oryzae), Trichoderma (eg, Trichoderma reesei (Trichoderma reesei), Clostridium (e.g. Clostridium thermocellum), Cellulomonas (e.g. Cellulomonas fimi) and Cellulomonas uda (Cellulomonas uda), Examples include microorganisms belonging to the genus Pseudomonas (eg, Pseudomonas fluorescence) and the like.

  Hereinafter, endoglucanases, cellobiohydrolases, and β-glucosidase will be described as representative cellulolytic enzymes, but the cellulolytic enzymes are not limited thereto.

  Endoglucanases are enzymes commonly referred to as cellulases, which cleave cellulose from the interior of the molecule to produce glucose, cellobiose, and cellooligosaccharides ("intramolecular cleavage"). There are five types of endoglucanases, which are called endoglucanase I, endoglucanase II, endoglucanase III, endoglucanase IV and endoglucanase V, respectively. These distinctions are amino acid sequence differences, but they are common in that they have an intramolecular cleavage effect on cellulose. For example, Trichoderma reesei-derived endoglucanase (in particular, endoglucanase II: EGII (eg, Patent Document 6)) can be used, but is not limited thereto.

  Cellobiohydrolase degrades from either the reducing end or the non-reducing end of cellulose to release cellobiose ("cellulose molecular terminal cleavage"). There are two types of cellobiohydrolases, which are called cellobiohydrolase I and cellobiohydrolase II, respectively. These distinctions are amino acid sequence differences, but they are common in that they have a cellulose molecular terminal cleaving action. For example, Trichoderma reesei-derived cellobiohydrolase (in particular, cellobiohydrolase II: CBHII (eg, Patent Document 6)) can be used, but is not limited thereto.

  [beta] -glucosidase is an exo-type hydrolase that cleaves glucose units from the non-reducing end of cellulose ("glucose unit cleavage"). β-glucosidase can cleave β1,4-glycosidic bonds between aglycones or sugar chains and β-D-glucose, and can hydrolyze cellobiose or cellooligosaccharides to produce glucose. β-glucosidase is a representative example of an enzyme capable of hydrolyzing cellobiose or cellooligosaccharides. One type of β-glucosidase is currently known and is called β-glucosidase 1. For example, Aspergillus acreatus-derived β-glucosidase (in particular, β-glucosidase 1: BGL1 (eg, non-patent document 7)) can be used, but is not limited thereto.

  For good hydrolysis of cellulose, enzymes with different cellulose hydrolysis modes may be combined. Enzymes that act in a variety of different cellulose hydrolysis modes can be combined, as appropriate, such as intramolecular cellulose cleavage, cellulose molecular termination, and glucose unit cleavage. Examples of enzymes with respective modes of hydrolysis include, but are not limited to endoglucanases, cellobiohydrolases, and β-glucosidases. The combination of enzymes with different modes of cellulose hydrolysis may for example be selected from the group consisting of endoglucanases, cellobiohydrolases, and β-glucosidases. Since it is desirable to be able to ultimately produce glucose, which is a constituent sugar of cellulose, it is preferable to include at least one enzyme capable of producing glucose. As enzymes capable of producing glucose, in addition to glucose unit cleaving enzymes (eg, β-glucosidase), endoglucanases can also produce glucose. For example, in yeast, β-glucosidase, endoglucanase, and cellobiohydrolase can be secreted or surface-displayed.

(Terminator DNA)
The polynucleotide comprising the secretion cassette or the surface display cassette may further comprise a terminator DNA.

  The terminator DNA should have terminator activity, and "terminator activity" refers to the activity to terminate transcription in the terminator region. The terminator only needs to have terminator activity, and can be excised from a cell, phage or the like carrying a desired terminator region using a restriction enzyme. If necessary, a DNA fragment of a terminator region can be obtained by amplifying a desired terminator region by PCR using a primer provided with a restriction enzyme recognition site or an insertion site of a cloning vector. Alternatively, desired terminator DNA may be chemically synthesized based on already known nucleotide sequence information of the terminator region.

  Examples of the terminator DNA include α-agglutinin terminator, ADH1 (aldehyde dehydrogenase) terminator, TDH3 (glyceraldehyde-3′-phosphate dehydrogenase) terminator, DIT1 terminator and the like.

(Construction of expression cassette)
In the present specification, an "expression cassette" refers to a DNA encoding a target protein and various regulatory elements for regulating the expression thereof so that the target protein can be expressed in a microorganism or cell of a host. A DNA sequence or polynucleotide linked in a functional manner. Here, "functionally linked" means that the expression cassette is such that the DNA encoding the target protein is expressed under the control of a promoter and optionally under the control of other regulatory elements. Alternatively, it means that each component contained in the expression vector is linked. Each component may be further linked including a sequence such as a linker between the elements as long as the target protein can be expressed.

  Examples of expression cassettes include secreted expression cassettes and surface-displayed expression cassettes as described below.

  The secretory type expression cassette includes a promoter DNA, a DNA encoding a secretory signal peptide of the present invention, and a DNA encoding a target protein (this expression cassette is also referred to as a secretory cassette).

  The surface display type expression cassette includes a promoter DNA, a DNA encoding a secretion signal peptide of the present invention, a DNA encoding a target protein, and a DNA encoding an anchor domain (this expression cassette is also referred to as a surface display cassette) .

  In the secretion cassette, a promoter DNA, a DNA encoding a secretion signal peptide, and a DNA encoding a target protein can be linked in a state where they can function in a host microorganism or host cell. The secretion cassette is constructed to contain, for example, the promoter DNA, the DNA encoding the secretion signal peptide of the present invention, and the DNA encoding the target protein in the order listed from 5 'to 3'. The secretion cassette may further comprise terminator DNA downstream of the DNA encoding the protein of interest.

  In the surface display cassette, a promoter DNA, a DNA encoding a secretory signal peptide, a DNA encoding a target protein, and a DNA encoding an anchor domain can be linked in a functional manner in a host microorganism or host cell. The surface layer display cassette includes, for example, in the order of 5 'to 3', promoter DNA, DNA encoding the secretion signal peptide of the present invention, DNA encoding the target protein, and DNA encoding the anchor domain. To be built. The surface display cassette may further comprise terminator DNA downstream of the DNA encoding the anchor domain.

  The synthesis and ligation of DNA containing various sequences can be performed by techniques that can be routinely used by those skilled in the art. For binding of each component, for example, DNA of each component to which a recognition sequence of a suitable restriction enzyme is imparted by PCR method is cleaved with the restriction enzyme and ligated using ligase or the like, or One-step It can carry out using isothermal assembly (nonpatent literature 8). By using these methods it is possible to cleave the correct secretion signal peptide and to express the active enzyme.

  From a plasmid containing a region (structural gene) encoding a target protein (eg, an enzyme such as endoglucanase, cellobiohydrolase, β-glucosidase, a fluorescent protein, or various antibodies), and an expression control sequence such as a promoter and a terminator Alternatively, it can be used by preparing an insert by excising it in a form suitable for the preparation of a vector.

  Alternatively, the expression cassette (secretion cassette and surface display cassette) of the present invention may contain a cloning site for inserting DNA encoding a target protein instead of including DNA encoding a target protein. . Such cloning sites are known in the art and, for example, multiple cloning sites can be utilized which include sites recognized by various restriction enzymes. In the case of an expression cassette containing such a cloning site, it becomes easy to insert the DNA encoding the target protein into the expression cassette via the cloning site.

(Expression vector)
The present invention provides an expression vector comprising the above expression cassette. The secretory expression vector of the present invention comprises the secretory expression cassette, and the surface display expression vector of the present invention comprises the surface display expression cassette. As used herein, the term "expression vector" refers to a vector into which a unit (expression cassette) for expression of a DNA encoding a target protein is inserted, including a DNA into which a DNA encoding a target protein is inserted. The expression vector may be a plasmid vector or may be an artificial chromosome. When yeast is used as a host, the form of a plasmid is preferred in that the preparation of the vector is easy and the transformation of yeast cells is easy. From the viewpoint of simplification of DNA acquisition, a shuttle vector of yeast and E. coli is preferred. If necessary, the vector may contain regulatory sequences (operators, enhancers, etc.). Such a vector has, for example, an origin of replication (Ori) of a 2 μm plasmid of yeast and an origin of replication of ColE1, a yeast selection marker (described below) and an E. coli selection marker (drug resistance gene etc.) ).

  Any known marker can be used as a yeast selection marker. For example, a drug resistance gene, a gene encoding auxotrophic marker gene (eg, imidazole glycerol phosphate dehydrogenase (HIS3), a gene encoding malate beta-isopropyl dehydrogenase (LEU2), O-acetylhomoserine O-acetylserine sulfide A gene encoding lylase (MET15), a gene encoding tryptophan synthase (TRP5), a gene encoding argininosuccinate lyase (ARG4), a gene encoding N- (5'-phosphoribosyl) anthranilate isomerase (TRP1), A gene encoding histidinol dehydrogenase (HIS4), a gene encoding orotidine-5-phosphate decarboxylase (URA3), dihydroorotic acid dehydrogenase And the gene encoding galactokinase (GAL1) and the gene encoding alpha-aminoadipate reductase (LYS2). For example, an auxotrophic marker gene (eg, HIS3, LEU2, URA3, MET15 deficient marker, etc.) can be preferably used.

(Preparation of transformed yeast)
The yeast used as a host is not particularly limited as long as it belongs to Ascomycetous yeast. For example, Saccharomyces (Saccharomyces), Kluyveromyces (Kluyveromyces), Candida (Candida), Pichia (Pichia), Schizosaccharomyces (Schizosaccharomyces), Hansenula (Hancenula), Klockera (Kloeckera), Examples include yeasts such as Schwanniomyces, Komagataella and Yarrowia.

  The yeast of the present invention can be obtained by introducing the above expression cassette or expression vector into a yeast serving as a host. The term "introduction" includes not only introduction into a host cell of a gene (DNA encoding a target protein) targeted for expression in an expression cassette or expression vector, but also expression in a host cell. The introduction method is not particularly limited, and known methods can be adopted. Typically, a method of transforming yeast using the expression vector of the present invention described above can be mentioned. The transformation method is not particularly limited, and publicly known methods such as calcium phosphate method, electroporation method, lipofection method, DEAE dextran method, lithium acetate method, transfection method such as protoplast method and microinjection method are used without limitation. it can. The introduced gene may be present in the form of a plasmid, or may be present in the form inserted into the yeast chromosome or in the form integrated by homologous recombination in the yeast chromosome.

  By introducing an expression vector containing a secretion cassette into yeast and obtaining a transformed yeast, a yeast that secretes a protein can be produced. By introducing an expression vector containing a surface layer display cassette into yeast to obtain a transformed yeast, a yeast that displays proteins on the cell surface can be produced.

  The yeast into which the above expression cassette or expression vector has been introduced can be transformed according to a conventional method into a trait by a yeast selection marker, an activity of a target protein of a bacterial cell (surface display type) or an activity of a target protein outside extracellular (secretory type) It can be selected as an indicator.

  In addition, it can be confirmed by a conventional method that the target protein is immobilized (cell surface display) on the cell surface of the transformed yeast obtained by introducing the expression vector containing the surface layer display cassette into yeast. For example, a method in which an antibody against this protein and a fluorescently labeled secondary antibody such as FITC or an enzyme-labeled secondary antibody such as alkaline phosphatase are allowed to act on a test yeast, an antibody against this protein and a biotin-labeled secondary antibody And the like, and a method of causing fluorescent labeled streptavidin to act further, and the like.

  Yeast can also be transformed to secrete or express on the surface of multiple proteins. In this case, each expression vector containing a gene expression cassette of a sequence encoding each of a plurality of types of proteins may be constructed, or a plurality of gene expression cassettes may be contained in one expression vector. For example, a vector pATP403 for simultaneous expression of three genes can be used (Non-patent Document 9).

  The transformed yeast of the present invention can be cultured under culture conditions generally applicable to yeast. Cultivation of transformed yeast can be appropriately carried out by methods known to those skilled in the art. The medium composition, the culture pH and the culture temperature can be appropriately set according to the nature of the yeast and the target protein. Cell density and culture time during culture may also be appropriately set according to the nature of the yeast and the target protein.

  Furthermore, according to the present invention, there is also provided a method of secretory production of a protein. This method can be carried out using the culture step described above for the secretory form of the transformed yeast. For example, proteins such as antibodies and enzymes can be secreted and produced by the method. If necessary, the step of recovering the production-containing fraction from the culture solution, and the step of purifying or concentrating it can also be carried out. Those steps and means required for them are appropriately selected by those skilled in the art.

  When the transformed yeast of the present invention is subjected to fermentation culture, culture conditions generally applied to the yeast can be appropriately selected and used. Typically, stationary culture, shaking culture, aeration stirring culture and the like can be used for culture for fermentation. The aeration conditions can be appropriately selected from anaerobic conditions, microaerobic conditions, aerobic conditions and the like. The medium composition, medium pH, culture temperature, cell density in culture and culture time, and subsequent recovery, purification, and concentration can be appropriately set.

  EXAMPLES Hereinafter, the present invention will be described by way of examples, but the present invention is not limited by these examples.

  The amino acid sequences of the SED1 secretion signal (SEQ ID NO: 2) and the CWP2 secretion signal (SEQ ID NO: 20) are derived from the signal peptide prediction program PSORT (http: // psort. Extracted based on the prediction by nibb.ac.jp/ (available as WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html)).

  Yeast Saccharomyces cerevisiae strain BY4741 (non-patent document 8) used in this example was obtained from Invitrogen.

  All PCR methods shown in this example were performed using KOD-Plus-Neo-DNA polymerase (manufactured by Toyobo Co., Ltd.).

  Gene transfer to all the yeasts shown in this example was carried out by the lithium acetate method.

Preparation Example 1: Preparation of Vector Plasmid Containing Various Expression Cassettes
Plasmids were prepared containing the following expression cassettes X1 to X19 respectively:
X1: SED1 promoter + glucoamylase secretion signal + BGL1 + SED1 anchor domain X2: SED1 promoter + SED1 secretion signal + BGL1 + SED1 anchor domain X3: SED1 promoter + MFα prepro + BGL1 + SED1 anchor domain X4: SED1 promoter + HKR1 secretion signal + BGL1 + SED1 anchor domain X1
X6: SED1 promoter + SED1 secretion signal + BGL1
X7: SED1 promoter + MFα prepro + BGL1
X8: SED1 promoter + HKR1 secretion signal + BGL1
X9: SED1 promoter + glucoamylase secretion signal + EGII + SED1 anchor domain X10: SED1 promoter + SED1 secretion signal + EGII + SED1 anchor domain X11: SED1 promoter + MFα prepro + EGII + SED1 anchor domain X12: TDH3 promoter + glucoamylase secretion signal + BGL1 + SED1 anchor domain X13: TDH3 Anchor domain X14: TDH3 promoter + MFα prepro + BGL1 + SED1 anchor domain X15: PGK1 promoter + glucoamylase secretion signal + BGL1 + SED1 anchor domain X16: PGK1 promoter + SED1 secretion signal + BGL1 + SED1 anchor domain X17: PGK1 promoter + MFα prepro + BGL1 + SED1
X19: CWP2 promoter + CWP2 secretion signal + BGL1
X20: Pichia pastoris -derived TDH1 promoter + MFα prepro + GFP
X21: Pichia pastoris -derived TDH1 promoter + glucoamylase secretion signal + GFP
X22: Pichia pastoris -derived TDH1 promoter + SED1 secretion signal + GFP

The nucleotide sequence and amino acid sequence of each component contained in the above expression cassette are as shown below:
SEQ ID NO: 1: Nucleotide sequence of a DNA encoding a secretion signal peptide of SED1 derived from Saccharomyces cerevisiae. SEQ ID NO: 2: Amino acid sequence of a secretion signal peptide of SED1 derived from Saccharomyces cerevisiae. SEQ ID NO: 3: Promoter of SED1 derived from Saccharomyces cerevisae. Nucleotide sequence of SEQ ID NO: 4: base sequence of DNA of a region of the coding region of SED1 anchor protein derived from Saccharomyces cerevisiae used as a SED1 anchor domain in the examples of the present invention excluding the initiation codon SEQ ID NO: 5 of the present invention Amino acid sequence of SED1 anchor protein from Saccharomyces cerevisiae (but not including the initiation methionine) used as a SED1 anchor domain in the examples SEQ ID NO: 6: derived from Rhizopus oryzae Nucleotide sequence of DNA encoding glucoamylase secretion signal peptide SEQ ID NO: 7: Amino acid sequence of glucoamylase secretion signal peptide from Rhizopus olize SEQ ID NO. 8: Nucleotide sequence sequence of DNA encoding MFα prepro derived from Saccharomyces cerevisiae : Amino acid sequence of MFα prepro derived from Saccharomyces cerevisiae SEQ ID NO: 10: Nucleotide sequence of DNA encoding secretory signal peptide of secretory protein HKR1 derived from Saccharomyces cerevisae SEQ ID NO 11: Secreted signal peptide of secretory protein HKR1 derived from Saccharomyces cerevisiae Amino acid sequence of SEQ ID NO: 12: base sequence of TDH3 promoter derived from Saccharomyces cerevisiae SEQ ID NO: 13: base sequence of PGK1 promoter derived from Saccharomyces cerevisae SEQ ID NO: 1 : Nucleotide sequence of DNA encoding .beta.-glucosidase 1 (BGL1) derived from Aspergillus acreatus SEQ ID NO: 15 amino acid sequence of .beta.-glucosidase 1 derived from Aspergillus acreatus 1 (BGL1) SEQ ID NO: 16: Endoglucanase II derived from Trichoderma reesei (EGII) SEQ ID NO: 17: Amino acid sequence of endoglucanase II (EGII) from Trichoderma reesei SEQ ID NO: 18: Nucleotide sequence of CWP2 promoter from Saccharomyces cerevisiae SEQ ID NO: 19: CWP2 secretion from Saccharomyces cerevisae Nucleotide sequence of DNA encoding signal peptide SEQ ID NO: 20: Amino acid sequence of CWP2 secretion signal peptide from Saccharomyces cerevisiae SEQ ID NO: 21: TDH1 promoter from Pichia pastoris Group SEQ SEQ ID NO: 22: Pichia pastoris sea mushroom Green 1 optimized in codon (mUkG1) of DNA encoding the nucleotide sequence SEQ ID NO: 23: amino acid sequence of the sea mushroom Green 1 (mUkG1)

  Hereinafter, preparation procedures of various plasmids used to obtain a transformed yeast strain used in this example will be described.

  The cell surface localization protein gene Sed1 derived from Saccharomyces cerevisiae is PCR-analyzed using the genome of Saccharomyces cerevisiae strain BY4741 as a template and the primer pair Sed1p-F (SEQ ID NO: 24) and Sed1ss-R (SEQ ID NO: 25). After amplification, a DNA fragment containing a promoter region and a secretion signal sequence was prepared. This fragment is expressed in vector plasmid pIBG-SGS (Auxotrophic marker gene HIS3 and BGL1 expression cassette (ie, SED1 promoter, secretory signal peptide sequence of Rhizopus oryzae derived glucoamylase, β-glucosidase 1 from Aspergillus acreatus (BGL1)) A surface expression vector having a cassette in which a coding region of the gene, a coding region of the SED1 gene, and a coding region of 445 bp downstream of the coding region of the α-agglutinin gene are arranged in this order: pIBG of non-patent document 10 The fragment amplified using -P (corresponding to -SS) as a template and the primer pair BGL1-F (SEQ ID NO: 26) and PRS-R (SEQ ID NO: 27) was ligated by One-step isothermal assembly. The resulting plasmid containing the expression cassette X2 was named pIBG-SSS.

  A fragment containing the MFα prepro leader sequence derived from Saccharomyces cerevisiae is expressed by pIUPGSBAAG (MFα prepro sequence and a vector for surface expression of α-amylase having a region on the 3 'side of the α-agglutinin gene by PCR method): Non-patent document 11) was used as a template and was prepared by amplification using primer pair MFa-F (SEQ ID NO: 28) and MFa-R (SEQ ID NO: 29). This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SGS as a template and the primer pair BGL1-F2 (SEQ ID NO: 30) and Sed1p-R (SEQ ID NO: 31) by one-step isothermal assembly. The resulting plasmid containing the expression cassette X3 was named pIBG-SMS.

  The secreted protein gene Hkr1 derived from Saccharomyces cerevisiae is amplified by PCR using the genome of Saccharomyces cerevisiae strain BY4741 as a template and the primer pair Hkr1ss-F (SEQ ID NO: 32) and Hkr1ss-R (SEQ ID NO: 33), A DNA fragment containing the secretory signal sequence was prepared. This fragment was ligated to a fragment amplified using the vector plasmid pIBG-SGS as a template and the primer pair BGL1-F3 (SEQ ID NO: 34) and Sed1p-R2 (SEQ ID NO: 35) by one-step isothermal assembly. The resulting plasmid containing the expression cassette X4 was named pIBG-SHS.

  Sequences other than the coding region of the SED1 gene of pIBG-SGS, pIBG-SSS, pIBG-SMS, and pIBG-SHS were used as a template for the respective plasmid as a template, and the primer pair PRS-BsrGI-F (SEQ ID NO: 36) and BGL1- Prepared by amplification using BsrGI-R (SEQ ID NO: 37). Each of these fragments was treated with BsrGI and circularized by the self ligation method. The resulting plasmids containing the expression cassettes X5, X6, X7 or X8 were named pIBG-SGsec, pIBG-SSsec, pIBG-SMsec, and pIBG-SHsec, respectively.

  The coding region of the Trichoderma reesei-derived endoglucanase II (EGII) gene was amplified by PCR using the pIEG-SGS (auxotrophic marker gene HIS3 and EGII expression cassettes (that is, the secretory signal of the SED1 promoter, Rhizopus oryzae-derived glucoamylase) Surface expression vector having a cassette in which the peptide sequence, the coding region of the EGII gene, the coding region of the SED1 gene, and the coding region of the α-agglutinin gene downstream of 445 bp are arranged in this order: expression cassette X9) It was prepared by amplifying using the primer pair EGII-F (SEQ ID NO: 38) and EGII-R (SEQ ID NO: 39), using pIEG-SS (Reference 10) as a template. This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SSS as a template and the primer pair Sed1a-F (SEQ ID NO: 40) and Sed1ss-R2 (SEQ ID NO: 41) by one-step isothermal assembly. The resulting plasmid containing the expression cassette X10 was named pIEG-SSS.

  The coding region of the EGII gene was prepared by PCR amplification using pIEG-SGS as a template and the primer pair EGII-F2 (SEQ ID NO: 42) and EGII-R (SEQ ID NO: 39). This fragment was ligated to a fragment amplified using the vector plasmid pIBG-SMS as a template and the primer pair Sed1a-F (SEQ ID NO: 40) and MFa-R2 (SEQ ID NO: 43) by One-step isothermal assembly. The resulting plasmid containing the expression cassette X11 was named pIEG-SMS.

  PIBG-TGS (Auxotrophic marker gene HIS3 and BGL1 expression cassette (ie, TDH3 promoter, secretion signal peptide sequence of Rhizopus oryzae-derived glucoamylase, BGL1) by PCR method for the promoter region of the gene TDH3 derived from Saccharomyces cerevisiae A surface expression vector having a cassette in which a coding region of the gene, a coding region of the SED1 gene, and a coding region of 445 bp downstream of the coding region of the α-agglutinin gene are arranged in this order: pIBG of Non-Patent Document 10 (Corresponding to -TS) as a template and prepared by amplification using a primer pair TDH3p-F (SEQ ID NO: 44) and TDH3p-R (SEQ ID NO: 45). This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SSS as a template and the primer pair Sed1ss-F (SEQ ID NO: 46) and PRS-R2 (SEQ ID NO: 47) by one-step isothermal assembly. The resulting plasmid containing the expression cassette X13 was named pIBG-TSS.

  The promoter region of the gene TDH3 derived from Saccharomyces cerevisiae is prepared by PCR using pIBG-TGS as a template and using the primer pair TDH3p-F (SEQ ID NO: 44) and TDH3p-R2 (SEQ ID NO: 48) did. This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SMS as a template and the primer pair MFa-F2 (SEQ ID NO: 49) and PRS-R2 (SEQ ID NO: 47) by One-step isothermal assembly. The resulting plasmid containing the expression cassette X14 was named pIBG-TMS.

  Using the promoter region of the gene PGK1 derived from Saccharomyces cerevisiae as a template by PCR, using pGK403 (a vector for protein expression having a promoter region and a terminator region of PGK1: non-patent document 12) as a template, primer pair PGK1p-F (SEQ ID NO: 50 And PGK1p-R (SEQ ID NO: 51) for amplification. This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SGS as a template and the primer pair GAss-F (SEQ ID NO: 52) and PRS-R3 (SEQ ID NO: 53) by One-step isothermal assembly. The resulting plasmid containing the expression cassette X15 was named pIBG-PGS.

  The promoter region of the gene PGK1 derived from Saccharomyces cerevisiae was prepared by PCR using pGK403 as a template and amplification using the primer pair PGK1p-F (SEQ ID NO: 50) and PGK1p-R2 (SEQ ID NO: 54). This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SSS as a template and the primer pair Sed1ss-F2 (SEQ ID NO: 55) and PRS-R3 (SEQ ID NO: 53) by one-step isothermal assembly. The resulting plasmid containing the expression cassette X16 was named pIBG-PSS.

  The promoter region of the gene PGK1 derived from Saccharomyces cerevisiae was prepared by PCR using pGK403 as a template and amplification using the primer pair PGK1p-F (SEQ ID NO: 50) and PGK1p-R3 (SEQ ID NO: 56). This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SMS as a template and the primer pair MFa-F3 (SEQ ID NO: 57) and PRS-R3 (SEQ ID NO: 53) by One-step isothermal assembly. The resulting plasmid containing the expression cassette X17 was named pIBG-PMS.

  The promoter region of the cell surface localized protein gene CWP2 derived from Saccharomyces cerevisiae is PCR by using the genome of Saccharomyces cerevisiae strain BY4741 as a template, and the primer pair Cwp2p-F (SEQ ID NO: 58) and Cwp2p-R (SEQ ID NO: 59) Prepared by amplification using This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SSsec as a template and the primer pair Sed1ss-F3 (SEQ ID NO: 60) and PRS-R4 (SEQ ID NO: 61) by one-step isothermal assembly. The resulting plasmid containing the expression cassette X18 was named pIBG-CSsec.

  The promoter region and secretory signal sequence of the cell surface localized protein gene CWP2 derived from Saccharomyces cerevisiae is PCR-mediated by using the genome of Saccharomyces cerevisiae strain BY4741 as a template, and the primer pair Cwp2p-F (SEQ ID NO: 58) and Cwp2ss-R (Sequence No. 58) Prepared by amplification using SEQ ID NO: 62). This fragment was ligated with a fragment amplified using the vector plasmid pIBG-SSsec as a template and the primer pair BGL1-F4 (SEQ ID NO: 63) and PRS-R4 (SEQ ID NO: 61) by One-step isothermal assembly. The resulting plasmid containing the expression cassette X19 was named pIBG-CCsec.

  The promoter region of the TDH1 gene from Pichia pastoris, the SpeI site, the MFα prepro leader sequence from Saccharomyces cerevisiae, the XhoI site, the green fluorescent protein (GFP) coding region, and the terminator region of the AOX1 gene from Pichia pastoris in this order The arranged fragments were gene-synthesized with sites for restriction enzymes HindIII and BamHI at both ends. In addition, the sea urchin mushroom green 1 (mUkG1) gene optimized to the codon of Pichia pastoris was used for GFP gene. This fragment was treated with HindIII and BamHI and ligated into similarly treated plasmid pUC19. The resulting plasmid was designated pmUkG1_MFα. Subsequently, the G418 resistance gene sequence is amplified by PCR using Life Technology's plasmid pPIC9K as a template and the primer pair G418r-BamHI-F (SEQ ID NO: 64) and G418r-EcoRI-R (SEQ ID NO: 65) Prepared by This fragment was treated with BamHI and EcoRI and ligated to the similarly treated plasmid pmUkG1_MFα. The resulting plasmid containing the expression cassette X20 was named pGmUkG1_MFα.

  The secretory signal peptide sequence of the lysozyme derived lysoamylase was sequenced by PCR using pIBG-SGS as a template and primer pair MFa-SpeI-F (SEQ ID NO: 66) and MFa-XhoI-R (SEQ ID NO: 67). Prepared by amplification. This fragment was treated with SpeI and XhoI and ligated into the plasmid pGmUkG1_MFα, which was treated in the same manner to remove the MFα prepro leader sequence. The plasmid containing the obtained expression cassette X21 was named pGmUkG1_GA.

  The secretory signal peptide sequence of SED1 derived from S. cerevisiae is PCR-based on the genome of S. cerevisiae BY4741 strain as a template and the primer pair Sed1ss-SpeI-F (SEQ ID NO: 68) and Sed1ss-XhoI-R (SEQ ID NO: 69) Prepared by amplification using. This fragment was treated with SpeI and XhoI and ligated into the plasmid pGmUkG1_MFα, which was treated in the same manner to remove the MFα prepro leader sequence. The resulting plasmid containing the expression cassette X22 was named pGmUkG1_SED1.

Preparation Example 2 Preparation of Various Transformed Yeasts
The following plasmids described in Preparation Example 1 (pIBG-SGS, pIBG-SSS, pIBG-SMS, pIBG-SHS, pIBG-SGsec, pIBG-SSsec, pIBG-SMsec, pIBG-SHsec, pIEG-SGS, pIEG-SSS, pIEG-SMS, pIBG-TGS, pIBG-TSS, pIBG-TMS, pIBG-PGS, pIBG-PSS, pIBG-PMS, pIBG-CSsec, and pIBG-CCsec) are treated with NdeI, and each strain is yeast Saccharomyces cerevisiae strain BY4741. The cells were subjected to (MATα his3 leu2 met15 ura3 strain) and transformed by the lithium acetate method. These transformed strains are respectively BY-BG-SGS strain, BY-BG-SSS strain, BY-BG-SMS strain, BY-BG-SHS strain, BY-BG-SGS strain, BY-BG-SSsec strain, BY -BG-SMsec strain, BY-BG-SHsec strain, BY-EG-SGS strain, BY-EG-SSS strain, BY-EG-SMS strain, BY-BG-TGS strain, BY-BG-TSS strain, BY- It is referred to as BG-TMS strain, BY-BG-PGS strain, BY-BG-PSS strain, BY-BG-PMS strain, BY-BG-CSsec strain, and BY-BG-CCsec strain.

  The following plasmids (pGmUkG1_MFa, pGmUkG1_GA, and pGmUkG1_SED1) described in Preparation Example 1 were treated with BsiWI, respectively provided to the yeast Pichia pastoris CBS7435 strain (wild strain), and transformed by the lithium acetate method. These transformants are referred to as PP-GFP-MFα strain, PP-GFP-GA strain, and PP-GFP-SED1 strain, respectively.

Test Example 1 Examination of β-Glucosidase Activity (BGL)
For surface-displayed strains, BGL activity (amount of activity per dry cell weight (U)) of the cells was measured, and for secreted strains, BGL activity in the medium (activity amount (U) per liter of medium) was measured. Examination of (beta) -glucosidase (BGL) activity was performed according to the following procedures.

The cells are transferred to 5 mL of SD medium (supplemented with leucine, methionine, and uracil), cultured at 30 ° C., 180 rpm for 18 hours (pre-culture), and then transferred to 50 mL of 1 × YPD medium (initial OD ₆₀₀ = 0). .05), cultured at 30 ° C., 150 rpm (main culture). The culture solution was collected each 24 hours after the start of the main culture, and the cells and the medium were separated by centrifugation at 1,000 g for 5 minutes.

The measurement of the β-glucosidase activity of the cells was performed as follows:
(1) Wash the cells twice with distilled water;
(2) 500 μl of reaction solution (composition: 100 μl of 10 mM pNPG (p-nitrophenyl-β-D-glucopyranoside) (final concentration 2 mM); 500 mM sodium citrate buffer (pH 5.0) 50 μl (final concentration 50 mM); distilled water 250 μL; and 100 μL of yeast suspension (final cell concentration 1 to 10 g wet cells / L)), and react at 500 rpm and 30 ° C. for 10 minutes;
(3) After completion of the reaction, 500 μL of ₃ M Na ₂ CO ₃ was added to stop the reaction; and (4) After centrifuging at 10,000 g for 5 minutes, the absorbance ABS ₄₀₀ at ₄₀₀ nm of the supernatant was measured. The amount of enzyme that liberates 1 μmol of pNP (p-nitrophenol) in 1 minute is 1 U.

  When measuring BGL activity in the culture medium, the above reaction solution was prepared as described above except that 100 μL of culture medium was added instead of the yeast suspension.

Test Example 2 Examination of Endoglucanase (EG) Activity
The EG activity (active amount per dry cell weight (U)) of the surface layer-displayed strain was determined according to the following procedure.

The cells are transferred to 5 mL of SD medium (supplemented with leucine, methionine, and uracil), cultured at 30 ° C., 180 rpm for 18 hours (pre-culture), and then transferred to 50 mL of 1 × YPD medium (initial OD ₆₀₀ = 0). .05), cultured at 30 ° C., 150 rpm (main culture). Forty-eight hours after the start of the main culture, each culture solution was collected, and the cells and the medium were separated by centrifugation at 1,000 g for 5 minutes.

Measurement of endoglucanase activity of the cells was performed as follows:
(1) Wash the cells twice with distilled water;
(2) Reaction solution 2500 μL (Composition: 1 tablet of Cerazyme C tablet (Megazyme); 500 mM sodium citrate buffer (pH 5.0) 250 μL (final concentration 50 mM); 2000 μL distilled water; and 250 μL yeast suspension (final 10 g wet cell / L)), let stand and react at 38 ° C. for 4 hours;
(3) After completion of the reaction, after centrifuging at 10,000 g for 5 minutes, the absorbance 590 nm of the supernatant ABS ₅₉₀ is measured.

Test Example 3: Examination of GFP Secretion Expression in Pichia pastoris
For the GFP-secreting strain of Kia pastoris, GFP fluorescence intensity in the medium was measured according to the following procedure.

  The cells were transferred to 500 μL of BMGY medium in a 96-well deep well plate, cultured at 30 ° C. and 1800 rpm for 18 hours (pre-culture), and then each preculture was placed in a 96-well deep well plate. The cells were transferred to 500 μL of BMGY medium, and cultured at 30 ° C. and 1800 rpm (main culture). Twenty-four hours after the start of the main culture, the 96-well deep well plate was centrifuged at 3,000 rpm for 5 minutes to obtain respective culture supernatants.

  100 μL of each culture supernatant was added to a 96-well black plate, and GFP fluorescence intensity (excitation wavelength: 485 nm: fluorescence wavelength: 510 nm) of each well was measured with EnVison 2104 Multilabel Reader (manufactured by Perkin Elmer).

(Example 1: Comparison of various secretion signals in β-glucosidase surface display)
In this example, various surface-displayed transformed yeasts (BY-BG-SGS strain, BY-BG-SSS strain, BY-BG-SMS strain) obtained by introducing a plasmid containing each expression cassette of X1 to X4 The BGL activity of the cells was measured for the strain and the BY-BG-SHS strain).

  The results are shown in FIG. The horizontal axis of FIG. 1 indicates the culture time (“time (hour)”), and the vertical axis indicates the β-glucosidase activity (active amount per dry cell weight (“U / g dry cell weight”)) Symbols in Fig. 1 are as follows: black circle, SED1 secretion signal (SED1); black diamond, glucoamylase secretion signal (GA) from Rhizopus oryzae; black triangle, MFα prepro (MFα); Secretion signal (HKR1).

  As shown in FIG. 1, the BGL surface-displayed transformed strain using the SED1 secretion signal can be produced by MFα prepro, which is often used for highly efficient secretion protein expression, or Rhizopus, which is often used for surface-displayed protein expression. It was found that even when compared with the transformed strain using the Oryze-derived glucoamylase secretion signal, it exhibited considerably higher surface layer BGL activity.

Example 2 Comparison of Various Secretion Signals in β-Glucosidase Secretion
In this example, various secretory transformed yeasts (BY-BG-SGsec strain, BY-BG-SSsec strain, BY-BG-SMsec strain) obtained by introducing a plasmid containing each expression cassette of X5 to X8 , And BY-BG-SHsec) were measured for BGL activity in the culture medium.

  The results are shown in FIG. The horizontal axis of FIG. 2 indicates the culture time (“time (hour)”), and the vertical axis indicates β-glucosidase activity (activity in the medium (“U / L”). The symbols in FIG. Black circles, SED1 secretion signal (SED1); closed diamonds, glucoamylase secretion signal (GA); closed triangles, MFα prepro (MFα); and closed squares, HKR1 secretion signal (HKR1).

  As shown in FIG. 2, BGL-secreted transformants using the SED1 secretion signal are MFα prepro, which is often used for highly efficient secretory protein expression, and Rhizopus olize, which is often used for surface-displayed protein expression. It was found that even when compared with a transformant using a glucoamylase secretion signal derived from the strain, it exhibited a considerably higher BGL activity.

(Example 3: Comparison of various secretion signals in endoglucanase surface display)
In this example, various surface-displayed transformed yeasts (BY-EG-SGS strain, BY-EG-SSS strain, and BY-EG-SMS strain) obtained by introducing a plasmid containing each expression cassette of X9 to X11 The EG activity of the cells was measured for each strain.

  The results are shown in FIG. The vertical axis in FIG. 3 indicates EG activity (absorbance measurement value (“ABS 590”). The bar graph in FIG. 3 sequentially from the left: Rhizopus ・ Oryzae-derived glucoamylase secretion signal (GA), MFαprepro (MFα) and SED1 secretion Represents the signal (SED1).

  As shown in FIG. 3, even when endoglucanase activity is used as an indicator, the surface-displayed transformant using the SED1 secretion signal is higher than the surface-displayed transformant using a commonly used secretion signal. It was observed to show secretion efficiency.

Example 4 Examination of Combination with Various Promoters
In this example, various surface-displayed transformed yeasts (BY-BG-TGS strain, BY-BG-TSS strain, and BY-BG-TMS strain) obtained by introducing a plasmid containing each expression cassette of X12 to X17. The BGL activity of the cells was measured for the BY-BG-PGS strain, the BY-BG-PSS strain, and the BY-BG-PMS strain).

  The results are shown in FIG. 4 (upper graph: TDH3 promoter; and lower graph: PGK1 promoter). The horizontal axis of each graph in FIG. 4 indicates the culture time ("time (hour)"), and the vertical axis indicates the β-glucosidase activity (the amount of activity per dry cell weight ("U / g dry cell weight") Symbols in Fig. 4 are as follows: closed square, SED1 secretion signal (SED1); closed diamond, glucoamylase secretion signal (GA) from Rhizopus oryzae; and closed triangle, MFα prepro (MFα) .

  As shown in FIG. 4, even when combined with a promoter other than the SED1 promoter, the strain using the SED1 secretion signal is as high as or higher than the surface-displayed transformant strain using the secretion signal that is often used conventionally. It was observed that the activity was shown stably. In particular, in the TDH3 promoter, the increase in activity was remarkable as in the case of the combination with the SED1 promoter shown in FIG.

Example 5 Examination of Combination with Various Promoters
In this example, various secretion transformed yeasts (BY-BG-CSsec strain and BY-BG-CCsec strain) obtained by introducing a plasmid containing each expression cassette of X18 and X19 in the medium BGL activity was measured.

  The results are shown in FIG. The horizontal axis of FIG. 5 indicates the culture time (“time (hour)”), and the vertical axis indicates the β-glucosidase activity (activity in the medium (“U / L”). The symbols in FIG. Black diamonds, CWP2 promoter plus SED1 secretion signal; and black squares, CWP2 promoter plus CWP2 secretion signal.

  As shown in FIG. 5, with respect to the CWP2 promoter, the secretory transformants used in combination with the SED1 secretion signal are higher than the secretory transformants used in combination with the CWP2 secretion signal derived from the same gene. It was observed to show BGL activity.

Example 6 Examination of Secretory Ability in Heterologous Yeast
In this example, a GFP-secreting transformant (PP-GFP-MFα strain, PP-GFP-GA strain) of yeast Pichia pastoris obtained by introducing a plasmid containing each expression cassette of X20, X21 and X22 And GFP-GFP-SED1 strain) were measured for GFP fluorescence intensity in the medium.

  The results are shown in FIG. The vertical axis in FIG. 6 indicates the relative value of the GFP fluorescence intensity in the culture medium of each strain when the GFP fluorescence intensity in the culture medium of the PP-GFP-MFα strain is 1. The bar graph in FIG. 6 represents, from left to right: MFα prepro (MFα), Rhizopus oryzae-derived glucoamylase secretion signal (GA), and SED1 secretion signal (SED1).

  As shown in FIG. 6, even when Pichia pastoris is used as a host, the secretory transformant using the SED1 secretion signal is considerably more than the secretory transformant using the secretory signal which is commonly used in the prior art. It was observed to show high GFP fluorescence intensity. From this result, it was shown that the secretion amount of protein can be improved more than before even when the SED1 secretion signal is a host of yeast species other than Saccharomyces cerevisiae.

  According to the present invention, various proteins such as enzymes can be efficiently secreted extracellularly or presented on the cell surface. Thereby, it is very useful for the efficiency improvement of the substance production using yeast, cost reduction, and promotion of the spread. As more specific application fields, it may be considered that the secretory production of proteins such as antibodies and enzymes, and the efficient production of chemical products from cellulosic biomass by the yeast having cellulolytic enzymes displayed on the cell surface, and cost reduction.

Claims (10)

An expression vector comprising the following (i), (ii) and (iii) in this order :
(I) SED1 promoter DNA,
(Ii) the following:
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2,
(B) a peptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having a secretory signal activity,
(C) An amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2 and represented by SEQ ID NO: 2 A peptide having an amino acid sequence having at least 90% sequence identity with and having a secretory signal activity,
(D) a peptide encoded by a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence represented by SEQ ID NO: 1 and having a secretory signal activity, and (e) consisting of the nucleotide sequence represented by SEQ ID NO: 1 A peptide which is hybridized with a complementary strand of DNA and encoded by a base sequence having at least 90% sequence identity to the base sequence represented by SEQ ID NO: 1, and which has a secretory signal activity,
DNA encoding any peptide selected from the group consisting of: (iii) DNA encoding the target protein, or a cloning site for inserting DNA encoding the target protein.   The expression vector according to claim 1, wherein (iii) is a DNA encoding a target protein. A transformed yeast into which the expression vector according to claim 2 has been introduced. A method for producing a yeast that secretes and produces a protein, comprising
SED1 promoter DNA; (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, (b) an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 2; (C) an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2; A peptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown and having secretory signal activity, (d) having at least 90% sequence identity with the base sequence represented by SEQ ID NO: 1 A complementary strand of DNA encoded by the nucleotide sequence and having a secretory signal activity, and (e) a nucleotide sequence represented by SEQ ID NO: 1 It encodes any peptide selected from the group consisting of a hybridizing peptide having a sequence identity of at least 90% with the nucleotide sequence shown in SEQ ID NO: 1 and having a secretory signal activity Introducing an expression cassette containing DNA; and DNA encoding a target protein in this order into the yeast to obtain a transformed yeast,
Method, including. A method of secretory production of a protein in yeast, comprising
Comprising culturing the prepared yeast by the method described in transformed yeast or claim 4 according to claim 3, method. An expression vector comprising the following (i), (ii), (iii) and (iv) in this order :
(I) SED1 promoter DNA,
(Ii) the following:
(A) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2;
(B) a peptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having a secretory signal activity,
(C) An amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2 and represented by SEQ ID NO: 2 A peptide having an amino acid sequence having at least 90% sequence identity with and having a secretory signal activity,
(D) a peptide encoded by a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence represented by SEQ ID NO: 1 and having a secretory signal activity, and (e) consisting of the nucleotide sequence represented by SEQ ID NO: 1 A peptide having a secretory signal activity, which is encoded by a base sequence that hybridizes with a complementary strand of DNA and has at least 90% sequence identity to the base sequence shown in SEQ ID NO: 1,
DNA encoding any peptide selected from the group consisting of
(Iii) a DNA encoding a target protein, or a cloning site for inserting a DNA encoding the target protein;
(Iv) DNA encoding an anchor domain. The expression vector according to claim 6 , wherein the anchor domain is a SED1 anchor domain. The expression vector according to claim 6 or 7 , wherein said (iii) is a DNA encoding a target protein. A transformed yeast into which the expression vector according to claim 8 has been introduced. A method for producing yeast which surface-displays a protein, comprising
SED1 promoter DNA; (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, (b) an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 2; (C) an amino acid sequence obtained by substituting, deleting or adding one or several amino acid residues to the amino acid sequence represented by SEQ ID NO: 2; A peptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown and having secretory signal activity, (d) having at least 90% sequence identity with the base sequence represented by SEQ ID NO: 1 A complementary strand of DNA encoded by the nucleotide sequence and having a secretory signal activity, and (e) a nucleotide sequence represented by SEQ ID NO: 1 It encodes any peptide selected from the group consisting of a hybridizing peptide having a sequence identity of at least 90% with the nucleotide sequence shown in SEQ ID NO: 1 and having a secretory signal activity DNA: DNA encoding a target protein; and an expression cassette comprising, in this order, DNA encoding an anchor domain is introduced into yeast to obtain a transformed yeast,
Method, including.

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